The invention relates to a method for determining an analyte in a sample and to a kit for use in the method.
Starting from the prior art, the method of the invention comprises the steps:
i. The sample is applied in a sample application zone (ASZ) on a flow matrix in which transport of components present in the sample may take place (transport flow). The flow matrix further comprises:
a) optionally an application zone (AR*Z) for a binding reactant (Reactant*=R*) which is analytically detectable,
b) a detection zone (DZ) which is located downstream of ASZ and exhibits another binding reactant (Capturer) firmly anchored to the matrix and in which a complex (signal complex) containing the Capturer and the analyte and/or the Reactant* is formed in the method.
ii. The flow is allowed to effect the transport of sample components.
iii. The signal complex is detected in the detection zone and the measured signal is used for the determination of the analyte.
The invention is primarily directed to the flow matrix which may be of the same type as those previously used in, for example, immunochromatography, see below.
Suitable binding reactants are those which participate in so-called affinity reactions, especially biospecific affinity reactions, and covalent binding reactions, especially exchange reactions between free thiol and reactive disulphide and other reactions between soft electrophiles and soft nucleophiles. Common biospecific affinity reactions are immunochemical, i.e. between antibody and antigen or hapten. Other types of bioaffine reactions are hybridization between complementary nucleic acids (including oligonucleotides), reaction between lectin and carbohydrate structure, between Ig(Fc)-structure and Ig(Fc)-binding protein, such as protein A or protein G, etc. The bioaffine reactions include the reaction between a biomolecule and a synthetically prepared ligand/capturer.
For the type of method in question, one talks about non-competitive methods, for example sandwich technique, and competitive methods. Sandwich technique usually means that an analytically detectable complex is formed in which the analyte binds to two bioaffine counterparts, one of which is analytically detectable and the other is Capturer. In common competitive variants, the analyte and an analytically detectable analyte analogue will compete for a limiting amount of bioaffine counterpart. As examples of two competitive variants may be mentioned those that use: a) competition between analyte and analyte analogue, which is labelled, for a limiting amount of ligand in the form of a firmly anchored Capturer, and b) competition between analyte and analyte analogue in the form of firmly anchored Capturer for a limiting amount of soluble and analytically detectable bioaffine counterpart.
For further information on previously used methodology within the technical field of the invention it is referred to U.S. Pat. No. 4,861,711 (Behringwerke), WO 88/08534 (Unilever). U.S. Pat. Nos. 5,120,643 and 4,740,468 (Abbott), EP-A-284,232 and U.S. Pat. No. 4,855,240 (Becton Dickinson) and WO 96/22532 (Pharmacia AB).
Heteroforms
Compounds which can compete for the binding to a counterpart via one of the above mentioned binding reactions. Heteroforms may be isoforms of proteins, e.g. isoenzymes etc. Within the term heteroforms are included inter alia different forms of bioaffine complexes which xe2x80x9cresemblexe2x80x9d each other by meeting the above definition. Examples are immunocomplexes where the antigen is the same but the antibody is of different class/subclass. See further under the title xe2x80x9cAnalytexe2x80x9d below.
Determination of whether two compounds are heteroforms to each other may be made in so-called inhibition tests.
Problems to be Solved by the Invention
The components of a sample that may affect or influence the signal that is to be detected in DZ can be divided into two main groups: a) the analyte and b) components which directly or indirectly disturb the detection. Directly disturbing components are those which interfere with the signal as such, for example fluorescent components in serum in case the complex is to be detected by fluorescence. Examples of indirectly disturbing components are heteroforms with regard to Capturer and/or an added bioaffine reactant R (for example R*). Other indirectly disturbing components, for example heterophilic antibodies, may be present in the original sample and interfere with the formation of the signal complex in DZ. In certain embodiments of the invention, ligands that are released from the separation zone of the invention may act disturbingly (see Example 1).
Problems with disturbing components in samples have often meant that for analytes that are present in low concentrations, the separation of disturbing components and the detection have been performed in different systems.
An example where after ion-exchange separation, analysis has been carried out either by immunological systems or by on-line measurement of an absorbing group (460 nm), is in the measurement of carbohydrate deficient transferrins (CDT=CD-transferrin=asialo-, monosialo- and disialo-transferrin). When CDT is present at a relatively high concentration (10xe2x88x929 M), both detection alternatives have been possible, but at lower concentrations of analyte, immunological measurement is required. The ion-exchange chromatography separation is controlled from an advanced and costly equipment, which requires specially educated personnel. Also the traditional immunological tests are expensive and require well-educated personnel.
The technique for immunological on-line measurement after a chromatographic separation step has been described by Afeyan et al. (Nature 358 (1992) 603-604) and Irth et al. (Anal. Chem. 14 (1995) 355-361). Its difficulties have been summarized by Krull et al. (LC-GC 15(7) (1997) 620-629).
Transport of whole cells into DZ may interfere with the signal from the detection complex. It is previously known to use flow matrices where the cells are captured mechanically (through filtration) in a denser pre-zone (Oudheusden et al., Ann. Clin. Biochem. 28 (1991) 55-59).
EP-A-696,735 discloses a chromatographic immunoanalytical system where, in order to extend the measuring range for the analyte, a predetermined amount of analyte-binding antibody has been immobilized in the sample application zone so that a certain amount of analyte is retained therein.
EP-A-702,233 discloses a chromatographic immunoanalytical system where, in a similar manner to that described in EP-A-696,735, a dilution effect of the sample is achieved by capturing a certain amount of analyte before it reacts with labelled reactant which is then detected in the detection zone.
WO 97/35205 discloses a chromatographic membrane for immunoanalysis having (i) a zone for the detection of labelled analyte-binding reactant which has not bound to the analyte, and (ii) a zone for the detection of the complex between analyte-binding reactant and the analyte. The relative amounts of unbound analyte-binding reactant and analyte: reactant complex gives a measure of the amount of analyte in the sample.
WO 94/06012 discloses an analytical test apparatus having a negative control zone placed before the analyte detection zone. The negative control zone has the function to indicate the presence in the sample of components that affect the analyte detection so that it becomes unreliable.
A first main object of the invention is to create a simple and rapid method that facilitates the determination of an analyte in the presence of disturbing components. A particular object is to avoid problems with disturbing components that are soluble or suspendable in liquid media of interest.
A second main object of the invention is more rapid and simpler determinations of individual heteroforms or combinations thereof, especially heteroforms, that exhibit peptide, carbohydrate or lipid structures, including various types of biologically active compounds. Among lipids are included steroids and other fat-soluble substances.
A third main object of the invention is to facilitate the measurement of analytes in the concentration range  less than 10xe2x88x927 M, particularly  less than 10xe2x88x929 M, especially for samples containing disturbing heteroforms of the analyte.
A fourth main object of the invention is to simplify the determination of individual heteroforms or combinations thereof in samples originating from biological materials.
A fifth main object of the invention is to provide more rapid and simpler evaluations of libraries of compounds, for example chemical libraries, such as combinatorial libraries.
A subobject of the above mentioned four main objects is to improve the possibilities of making determinations in field environment (usually semi-quantitatively) as well as in advanced laboratories (with the possibility of accurate quantification).
The Invention
The above mentioned objects may be achieved with the method mentioned in the introductory part herein, if the flow matrix contains one or more separation zones (SZ) between ASZ and DZ, which should permit at least one component, capable of influencing the signal from the signal complex in DZ, to be retarded/separated. This should take place in SZ by means of the ligand interactions mentioned below, which can be reversible or irreversible. The component may be either a disturbing component or the analyte. If the component is not an analyte, the retardation means that the component (or components) migrates more slowly than the analyte through SZ or is bound irreversibly to SZ and thereby is prevented from reaching DZ such that the detection of analyte in DZ essentially will not be disturbed by the component (or components) in question. Usually, this means that there should be a sufficient amount of ligand for substantially all of the disturbing component or components in the sample to be affected. xe2x80x9cSubstantially allxe2x80x9d depends on the relative concentrations of the component(s), but usually means that at least 90%, preferably at least about 95%, and more preferably at least 99% of the disturbing component(s) are retarded or captured in the separation zone. The component may be the analyte if it is desired to study the capability of one or more ligands to bind the analyte. In this case such a ligand is immobilized in the separation zone.
The choice of retarding structure/ligand in the separation zone is determined by the components that are retarded. The retardation may be based on various more or less specific interactions between the ligand structure and the component(s) to be retarded; see below under the title xe2x80x9cSeparation zonexe2x80x9d. After the passage of SZ, the analyte will migrate with the transport flow to the detection zone (DZ), in which a complex containing the Capturer and the analyte and/or R* are formed.
In those cases where it is intended to retard one or more disturbing components, the formation of signal complexes will take place in the absence thereof. The detection of signal complexes in DZ may be taken as a qualitative or quantitative measure of the analyte.
In those cases where it is intended to retard the analyte, the point of time for the formation of a signal complex will be changed, or, if the analyte-ligand binding in SZ is irreversible, the formation of a signal complex may be completely inhibited. The formation of a signal complex in DZ will be a measure of the capability of the analyte to bind to the ligand in SZ.